Microcystin-LR and okadaic acid belong to a family of natural toxins which localize to the liver, inhibit[unreadable] the serine-threonine protein phosphatases PP1 and PP2A, inducing severe detrimental effects on the[unreadable] liver. These compounds are considered acute hepatotoxins and carcinogens. Little information with[unreadable] respect to the metabolism of these two toxins is available. While glutathione and cysteine conjugates of[unreadable] microcystin-LR have been identified in vertebrates and invertebrates, no phase I metabolites have been[unreadable] identified. Recent reports suggest that okadaic acid is metabolically activated to genotoxic products, yet[unreadable] no metabolites of okadaic acid have been identified in vertebrates. The long-term goal of this research is[unreadable] to develop a thorough understanding of the metabolic fate of the two hepatotoxins and to understand how[unreadable] and if xenobiotic metabolizing enzymes are regulated by these environmental toxins and how factors such[unreadable] as age, gender cytochrome P450 (CYP) polymorphisms, tobacco use, diet, and other environmental[unreadable] factors will influence toxicity by affecting the expression and activity of xenobiotic metabolizing enzymes[unreadable] and hence the profile metabolites formed. The objective of this proposal is to generate human metabolites[unreadable] of microcystin-LR and okadaic acid synthetically and enzymatically, using human recombinant xenobiotic[unreadable] metabolizing enzymes, and to create an "average" metabolite profile for each toxin derived from human[unreadable] primary hepatocytes. We will also examine the effect of parent toxins and individual metabolites on a[unreadable] human liver cell line, HepG2. We will further evaluate the influence of the parent toxins and selected[unreadable] metabolites on gene expression in BALB/c mice. The central hypothesis of the proposed research is that[unreadable] microcystin-LR and okadaic acid may be metabolized by xenobiotic metabolizing enzymes, and that[unreadable] metabolites diversify the effects of exposure to the parent toxins by exhibiting different biological activities[unreadable] in their respective target organs. Preliminary data indicates that microcystin-LR is metabolized by CYP1A2[unreadable] and that exposure to microcystin-LR induces expression of CYP1A2. This hypothesis will be tested by[unreadable] pursuing four specific aims: 1) synthesize, isolate and characterize metabolites of MC-LR and OA using a[unreadable] combination of enzymatic and chemical synthesis; 2) incubate MC-LR and OA with pooled human[unreadable] primary hepatocytes to generate an "average" metabolite profile; 3) Perform gene expression/ microarray[unreadable] experiments in mice with MC-LR, OA and selected MC-LR and OA metabolites; 4) Evaluate cytotoxicity[unreadable] and genotoxicity of individual metabolites using human liver HepG2 cell line. This research is innovative[unreadable] because a systematic analysis of MC-LR and OA metabolites and their toxicities has not been undertaken.[unreadable] This work is significant because diet, environment, gender, age, health, prior exposure to xenobiotics[unreadable] and genetic polymorphisms in liver xenobiotic metabolizing enzymes in humans will influence metabolism[unreadable] and susceptibility in different populations. This work will immediately link a xenobiotic metabolizing[unreadable] enzyme, such as a cytochrome P450 (CYP) with a specific metabolite and will evaluate the toxicity of[unreadable] individual metabolites.